Saltatory organelle movement is a ubiquitous form of intracellular motility, and participates in important physiological functions such as fast axonal transport in neurons. Yet very little is known about the molecular mechanism of saltatory movement. Many experiments that might shed light on the nature of this mechanism have not been performed because the permeability barrier formed by the plasma membrane of living cells excludes most appropriate experimental probes. To circumvent this plroblem, I have developed a method for permeabilizing fibroblasts while sustaining saltatory movement. The cells are treated with Brij 58, a nonionic detergent, in a buffered medium that contains ATP, Mg2+, EGTA, and polyethylene glycol. After lysis saltatory movements continue at a reduced level for 20-30 minutes, and are accessable to experimentation. Two important new findings from my preliminary studies with this system are: 1) saltatory movement is rapidly and reversibly inhibited by 100 MuM vanadate, suggesting that a dynein-like molecule might be involved in the mechanism, and 2) raising free CA++ to 1 MuM rapidly inhibits the movement. Factors that affect saltatory movement in permeabilized cells will be explored systematically, including the dependence on ATP concentration, pH, and divalent cations. The inhibitory effect of CA++ will be studied in detail. The possible involvement of cytoskeletal proteins that are important in other types of cell motility will be tested with appropriate probes. As well as continuing to study of the effects of vanadate and other inhibitors of dynein. I will examine the effects of probes that may interfere with actomyosin-dependent mechanisms, such as phalloidin, N-ethylmaleimide-modified heavy meromyosin, DNAase I, and cytochalasins. The role of microtubules will be examined using antimitotic drugs, taxol, and anti-tubulin antibodies. The effects on saltatory movement of a monoclonal antibody against intermediate filaments will also be tested. AVEC-DIC and video intensified immunofluorescence microscopy will be used to determine whether saltating organelles move along microtubules or other cytoskeletal fibers. This new permeabilized cell model provides a promising approach for analyzing the mechanism of saltatory movement.